CN113122502A

CN113122502A - Enhanced CART cell for promoting solid tumor infiltration and preparation method and cell medicine thereof

Info

Publication number: CN113122502A
Application number: CN201911415357.5A
Authority: CN
Inventors: 张娜; 杜冰; 刘小红; 吴诗佳; 席在喜
Original assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Current assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-16
Also published as: WO2021136538A1; CN115003700A

Abstract

The invention discloses an enhanced CART cell for promoting solid tumor infiltration, a preparation method thereof and a cell medicine, and relates to the technical field of CART cells. The CART cells disclosed herein contain a first nucleic acid sequence and a second nucleic acid sequence. The CART cell has higher infiltration capacity on tumor tissues of solid tumors, has stronger killing capacity on tumor cells, has wide clinical application prospect, and provides a new treatment idea and strategy for treating the solid tumors by using the CART cell.

Description

Enhanced CART cell for promoting solid tumor infiltration and preparation method and cell medicine thereof

Technical Field

The invention relates to the technical field of CART cells, in particular to an enhanced CART cell for promoting solid tumor infiltration, a preparation method thereof and a cell medicine.

Background

Adoptive T cell transfer (ACT) is currently the most promising approach to immunotherapy, and CD-19 specific CAR-T cells can show complete remission in the treatment of relapsing refractory acute lymphoblastic leukemia, with the main therapeutic procedure: the patient's own T cells (or T cells from allogeneic donors) are first isolated, then activated and genetically modified to obtain chimeric antigen receptor T cells (CAR-T), and finally returned to the patient. Chimeric antigen receptors are formed by linking an extracellular antigen recognition domain (usually an antibody single chain variable fragment scFv) to an intracellular signaling domain (the CD3 zeta chain of the T cell receptor or the simultaneous introduction of one or more costimulatory signals such as CD28 and 4-1BB), the extracellular portion of which confers the ability of T cells to recognize specific antigens. Can cross MHC restriction, can stimulate T cell proliferation through a signal transduction structural domain after being directly combined with an antigen recognized by the antigen, simultaneously activates the cytotoxicity of the T cell and promotes the secretion of cytokines, finally eliminates the cells with the antigen, and has better specificity and persistence.

Although CAR-T cells have been shown to be effective in treating hematological tumors, they still face challenges in treating solid tumors and the therapeutic effect is not ideal.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The CART cell provided by the invention has higher infiltration capacity on tumor tissues of solid tumors, improves the killing capacity on the tumor cells, has a wide clinical application prospect, and provides a new treatment idea and strategy for treating the solid tumors by using the CART cell.

The invention is realized by the following steps:

in a first aspect, embodiments of the invention provide an enhanced CART cell that promotes infiltration of a solid tumor, the CART cell comprising a first nucleic acid sequence and a second nucleic acid sequence;

the first nucleic acid sequence comprises a first coding sequence encoding a chimeric antigen receptor whose antigen-binding domain is capable of targeting the solid tumor;

the second nucleic acid sequence comprises a second coding sequence encoding a LIGHT protein and a signal peptide coding sequence located upstream of the second coding sequence;

the second coding sequence is expressed from a constitutive promoter.

The inventor of the present invention analyzes that one of the main reasons why CART cells are not ideal for treating solid tumors at present is that: the special vasculature and matrix barrier of solid tumors prevents T cell infiltration. Abnormal blood vessels with tumor growth promoting function can make T cells infiltrate into specialized High Endothelial Venules (HEV) to lose function, and the HEV with incomplete function can reduce infiltration of T cells. Meanwhile, the fibrous structure induced by tumor-associated stromal cells (CASCs) influences the infiltration and function of T cells, and the Cardioma-associated fibroplasts (CAFs) are used as a main subset of the CASCs and can also inhibit the function of the T cells by secreting cytokines such as TGF-beta and the like.

LIGHT proteins (macromolecules to lymphoxins, induibile, microorganisms With HSV glycoprotein D for HVEM, expressed by T Lymphocyte, TNFSF14) are members of the TNF superfamily of cytokines, an inducible inflammatory cytokine. The CART cell provided by the embodiment of the invention utilizes a constitutive promoter to drive the LIGHT protein to secrete and express in a large scale continuously, namely the LIGHT protein is over-expressed, when the CART cell contacts a tumor cell, the LIGHT protein which is secreted and expressed enables the infiltration capacity of the CART cell to a solid tumor to be effectively improved, and further the capacity of the CART cell to kill the tumor cell is improved, the CART cell has a wide clinical application prospect, and the invention provides a new treatment idea and strategy for treating the solid tumor by using the CART cell.

In alternative embodiments, the second nucleic acid sequence is located downstream of the first nucleic acid sequence, the constitutive promoter is located upstream of the first nucleic acid sequence, and both the first coding sequence and the second coding sequence are driven to be expressed by the constitutive promoter.

The first coding sequence and the second coding sequence are placed under the same constitutive promoter to be driven to express, and the first nucleic acid sequence and the second nucleic acid sequence are placed together, namely, are located on the same DNA chain, so that the length of the whole nucleic acid sequence can be reduced, the genome loading of the CART cell is reduced, and the transformation of the CART cell is facilitated. Of course, in other embodiments, the first coding sequence and the second coding sequence may be expressed from two separate promoters; even if the first nucleic acid sequence and the second nucleic acid sequence are placed on different DNA strands or placed on the same DNA strand with other nucleic acid sequences in between, CART cells having these and similar situations are also within the scope of the present invention.

In alternative embodiments, the constitutive promoter is selected from any one of the elongation growth factor-1 α (EF-1 α), the early Cytomegalovirus (CMV) promoter sequence, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, the actin promoter, the myosin promoter, and the heme and creatine kinase promoters.

However, the constitutive promoter of the present invention is not limited to the above promoter, and other types of promoters used to drive the expression of LIGHT protein are also within the scope of the present invention.

In alternative embodiments, the constitutive promoter is EF-1 α.

The constitutive promoter of the present invention can be selected according to practical requirements, and includes but is not limited to EF-1 alpha, as long as it can drive the LIGHT protein to be expressed in large quantities.

The term "mass expression" as used herein means that the amount of LIGHT protein secreted and expressed by CART cells is higher under the drive of the constitutive promoter than without the drive of the constitutive promoter.

In alternative embodiments, the LIGHT protein is a LIGHT full-length protein or a functional fragment thereof.

The LIGHT protein can be membrane-bound, intracellular free form, extracellular cleavage form, or other various active fragments, as long as it is selected from the LIGHT full-length protein and has an activity of increasing the killing of tumor cells by CART cells.

In alternative embodiments, the LIGHT protein is a soluble fragment of the full-length LIGHT protein.

In alternative embodiments, the LIGHT protein has the amino acid sequence shown in SEQ ID No. 15.

The soluble LIGHT (SEQ ID NO.15) lacking the intracellular segment and the transmembrane region still has normal biological functions, cancer cells constitutively expressing the soluble LIGHT cannot form tumors, and the ability of the LIGHT to kill tumor cells is stronger under the synergistic effect of IFN gamma, so that the tumor killing ability of CART cells can be improved.

In alternative embodiments, the LIGHT protein is fused to a VTP polypeptide. That is, the second coding sequence encodes a LIGHT protein and a VTP polypeptide.

In alternative embodiments, the amino acid sequence of the VTP polypeptide is set forth in SEQ ID No. 17.

The VTP polypeptide can target abnormal tumor blood vessels, and can play a role in targeted transportation by fusing the VTP polypeptide, so that LIGHT protein is brought to the abnormal tumor blood vessels, thereby promoting the normalization of the abnormal tumor blood vessels, promoting the formation of lymphoid structures at tumor parts to guide the aggregation and activation of the CART cells, mobilizing autoimmune function and remodeling the inhibition effect of a tumor microenvironment, further changing the effect limitation of the CART cells caused by the incapability of penetrating tumor tissues and immunosuppression, enhancing the activity of the CAR-T cells against solid tumors, and greatly improving the effect of the CART cells in the treatment of the solid tumors.

In alternative embodiments, the signal peptide encoded by the signal peptide coding sequence is selected from a membrane-integrated signal peptide or a secreted signal peptide.

In alternative embodiments, the membrane-integrated signal peptide is selected from any one of the CD8 signal peptide, CD28 signal peptide, GM-CSF signal peptide, CD4 signal peptide, and CD137 signal peptide.

In alternative embodiments, the secretory signal peptide is selected from any one of an IgG signal peptide and a cytokine signal peptide.

Preferably, the base sequence of the signal peptide coding sequence is shown in SEQ ID NO. 13.

In alternative embodiments, the chimeric antigen receptor further has a transmembrane domain and a costimulatory signaling region.

In alternative embodiments, the transmembrane domain is selected from the transmembrane domains of at least one of the following protein molecules: CD5, CD28, CD137, CD3 epsilon, CD154, CD45, CD4, CD9, CD37, CD16, CD33, CD22, CD134, and CD8 alpha.

In an alternative embodiment, the transmembrane domain is a CD8a transmembrane domain.

In alternative embodiments, the costimulatory signaling region comprises the intracellular domain of at least one of the following costimulatory molecules: OX40, CD3 γ, CD3 δ, CD134, CD5, CD79a, CD137, ICD3 ε, CD154, CD22, CD66d, CD2, CD28, CD4, CD5, CD79b, COS, 4-1BB, and CD3 ζ.

In alternative embodiments, the costimulatory signaling region comprises the intracellular costimulatory element of 4-1BB and the intracellular domain of CD3 ζ.

In alternative embodiments, the solid tumor is selected from any one of liver cancer, head and neck cancer, melanoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, prostate cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma and breast cancer.

In alternative embodiments, the solid tumor is prostate cancer;

it should be noted that the solid tumors of the present invention include, but are not limited to, liver cancer, head and neck cancer, melanoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, prostate cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma and breast cancer, and are also within the scope of the present invention for other types of solid tumors.

In alternative embodiments, the antigen binding domain is capable of targeting a specific membrane antigen of prostate cancer, the antigen binding domain being selected from any one of Fab, Fab ', F (ab') 2 and scFv.

In alternative embodiments, the antigen binding domain is an scFv.

It should be noted that the antigen binding domain in the embodiments of the present invention may be selected according to the type of solid tumor to be treated, and any scFv as the antigen binding domain is within the scope of the present invention.

In alternative embodiments, the amino acid sequence of the light chain variable region of the antigen binding domain is set forth in SEQ ID No.3, the amino acid sequence of the heavy chain variable region of the antigen binding domain is set forth in SEQ ID No.7, and the amino acid sequence of the hinge region between the heavy chain variable region and the light chain variable region of the antigen binding domain is set forth in SEQ ID No. 5.

The antigen binding domain composed of SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7 is prostate specific membrane antigen and can target prostate cancer.

The first nucleic acid sequence and the second nucleic acid sequence are not particularly limited as long as they encode the corresponding proteins, and those skilled in the art can easily obtain the nucleic acid sequences encoding the proteins on the basis of the clear protein sequences, and therefore, it is within the scope of the present invention that the first nucleic acid sequence and the second nucleic acid sequence can encode the corresponding proteins regardless of the specific nucleotide sequence changes.

In a second aspect, embodiments of the invention provide methods of making a reinforced CART cell for promoting solid tumor infiltration according to any one of the preceding embodiments, comprising: allowing the target T cell to contain the first nucleic acid sequence and the second nucleic acid sequence.

It should be noted that, in addition to the disclosure of the above CART cells, those skilled in the art can easily prepare the above CART cells by using the conventional techniques in the art, and therefore, it is within the scope of the present invention to prepare the above CART cells by any method.

In a third aspect, the embodiments of the present invention provide a cell drug for treating solid tumor, which contains the enhanced CART cell for promoting solid tumor infiltration according to any one of the previous embodiments as an active ingredient.

It should be noted that the CART cell provided by the present invention can be used alone as an active drug for treating a solid tumor, and can also be used in combination with other active drugs for treating a solid tumor, and therefore, it is within the scope of the present invention to use the CART cell provided by the present invention in combination with other anti-tumor drugs.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram of the structure of expression elements contained in the first nucleic acid sequence and the second nucleic acid sequence in example 1; in the figure, A is a schematic diagram of the structure of a first nucleic acid sequence, and B is a schematic diagram of the structure in which a first nucleic acid and a second nucleic acid are linked.

FIG. 2 is a schematic diagram of the structure of CJ-PSMA-LIGHT-VTP plasmid vector backbone.

FIG. 3 is a schematic diagram of the structure of the lentiviral packaging helper plasmid pMD2. G.

FIG. 4 is a schematic diagram of the structure of the lentiviral packaging helper plasmid psPAX 2.

FIG. 5 shows the results of the positive rate test of 293T cells infected by different viruses in example 1.

FIG. 6 is the CAR positivity test results for different CARTs in example 1.

FIG. 7 is a graph of LIGHT expression levels for the different CARTs in example 1.

Figure 8 is the killing efficiency results for different CART at different effective target ratios.

FIG. 9 shows the results when E: T is 1: 1, the detection result of the LIGHT content secreted by different CARTs.

FIG. 10 is a graph showing the proliferation of different CARTs in the same culture system over two weeks.

FIG. 11 is a graph of in vivo imaging of tumor size in mice injected with PSMA-CART or PSMA-CART + hLIGHT.

FIG. 12 is the size of the tumor visualized in mice treated with PSMA-CART or PSMA-CART + hLIGHT.

FIG. 13 is the in vivo imaging of tumor size in mice after PBS or hLIGHT injection.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example illustrates prostate cancer, and a CART cell that specifically targets prostate cancer and secretes and expresses LIGHT-VTP protein was constructed.

1, constructing a plasmid vector containing an expression chimeric antigen receptor and a LIGHT-VTP expression cassette, wherein the structure and the position relation of each element on the expression cassette are referred to as figure 1, and the skeleton of the plasmid vector is referred to as figure 2.

The method comprises the following specific steps:

a first nucleic acid sequence expressing a chimeric antigen receptor targeting a prostate specific membrane antigen was synthesized by kasey corporation, the first nucleic acid sequence (PSMA-CAR) comprising: the CD8 alpha signal peptide, the PSMA single-chain antibody heavy chain variable region, the Linker1, the PSMA single-chain antibody light chain, the CD8 hinge region, the CD8 alpha transmembrane domain, the 4-1BB intracellular costimulatory element and the CD3 zeta intracellular domain (A in figure 1) are connected in sequence, and a Kozac sequence and a corresponding enzyme cutting site are introduced into the forefront. The first nucleic acid sequence was transferred to a plasmid empty vector (lentiviral transfer vector of Carl June (CJ) team) using XbaI and SalI double digestion, and after enzymatic ligation, the chimeric antigen receptor expression vector CJ-PSMA-CAR was obtained as a control plasmid. Further, using CJ-PSMA-CAR plasmid as initial plasmid, adding a second nucleic acid sequence for expressing LIGHT-VTP, wherein the first nucleic acid sequence and the second nucleic acid sequence are connected by P2A, and the second nucleic acid sequence (LIGHT-VTP) sequentially comprises: secretion Signal Peptide (SP), LIGHT-VTP (B in FIG. 1). The resulting plasmid was named CJ-PSMA-LIGHT-VTP plasmid (see FIG. 2 for structure).

Wherein, the sequences of the elements of the first expression cassette for expressing the chimeric antigen receptor targeting the prostate specific membrane antigen are as follows:

the base sequence of the CD8 alpha signal peptide (CD8 alpha Leader) is shown as SEQ ID NO. 1:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg。

the base sequence of the PSMA single-chain antibody light chain variable region (PSMA-ScFv VL) is shown in SEQ ID NO. 2:

gacatcgtgatgacccagtccccctcctccctgtctgcctccgtgggcgacagagtgaccatcacatgcaaggcctcccaggattgtggcaccgccgtggactggtatcagcagaagcctggcaaggcccctaagctgctgatctactgggcctccaccagacacaccggcgtgcctgacagattcaccggctccggctctggcaccgacttcaccctgaccatctccagcctgcagcctgaggacttcgccgactacttctgccagcagtacaactcctaccctctgaccttcggcggaggcaccaagctggaaatcaaa；

the amino acid sequence of the PSMA single-chain antibody light chain variable region is shown in SEQ ID NO. 3:

DIVMTQSPSSLSASVGDRVTITCKASQDCGTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFT GSGSGTDFTLTISSLQPEDFADYFCQQYNSYPLTFGGGTKLEIK。

the base sequences of the Linker of the PSMA-ScFv VL and the PSMA-ScFv VH are shown in SEQ ID NO. 4:

ggcggaggcggatcaggtggtggcggatctggaggtggcggaagc；

the amino acid sequence of Linker is shown in SEQ ID NO. 5:

GGGGSGGGGSGGGGS。

the base sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO. 6:

gaagtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgcctccgtgaagatctcctgcaagacctccggctacaccttcaccgagtacaccatccactgggtgaaacaggcctccggcaagggcctggaatggatcggcaacatcaaccctaacaacggcggcaccacctacaaccagaagttcgaggaccgggccaccctgaccgtggacaagtccacctccaccgcctacatggaactgtcctccctgcggtctgaggacaccgccgtgtactactgcgccgctggctggaacttcgactactggggccagggcaccacagtgacagtctcgagc；

the amino acid sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO. 7:

EVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIHWVKQASGKGLEWIGNINPNNGGTTYNQKFEDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSS。

the base sequence of the CD8 hinge region (CD8 hinge) is shown as SEQ ID NO. 8:

accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat。

the base sequence of the CD8 alpha transmembrane domain (CD8a-TM) is shown as SEQ ID NO. 9:

atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc。

the base sequence of the intracellular costimulatory element of 4-1BB is shown in SEQ ID NO. 10:

aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg。

the nucleotide sequence of the intracellular domain of CD3 ζ is shown in SEQ ID NO. 11.

agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc。

The base sequence of P2A is shown in SEQ ID NO. 12:

gccacaaacttctctctgctaaagcaagcaggtgatgttgaagaaaaccccgggcct。

the base sequence of the secretion Signal Peptide (SP) is shown as SEQ ID NO. 13:

atggaaactgatactttgctgctctgggtcctgctgctgtgggtccctggatcaacgggggac。

the base sequence of LIGHT is shown in SEQ ID NO. 14:

ggagagatggtcacccgcctgcctgacggacctgcaggctcctgggagcagctgatacaagagcgaaggtctcacgaggtcaacccagcagcgcatctcacaggggccaactccagcttgaccggcagcggggggccgctgttatgggagactcagctgggcctggccttcctgaggggcctcagctaccacgatggggcccttgtggtcaccaaagctggctactactacatctactccaaggtgcagctgggcggtgtgggctgcccgctgggcctggccagcaccatcacccacggcctctacaagcgcacaccccgctaccccgaggagctggagctgttggtcagccagcagtcaccctgcggacgggccaccagcagctcccgggtctggtgggacagcagcttcctgggtggtgtggtacacctggaggctggggagaaggtggtcgtccgtgtgctggatgaacgcctggttcgactgcgtgatggtacccggtcttacttcggggctttcatggtg；

the amino acid sequence of LIGHT is shown in SEQ ID NO. 15:

GEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV。

the base sequence of VTP is shown in SEQ ID NO. 16:

ggcggcggctgccggggccggagaagcaccggc。

the amino acid sequence of VTP is shown in SEQ ID NO. 17: GGGCRGRRSTG are provided.

2 construction of viruses expressing chimeric antigen receptor and LIGHT-VTP

The method comprises the following steps: the CJ-PSMA-CAR and CJ-PSMA-LIGHT-VTP plasmids as well as the lentiviral packaging helper plasmids pMD2.G (see FIG. 3) and psPAX2 (see FIG. 4) were amplified using E.coli, and the plasmids were extracted and then subjected to agarose gel electrophoresis and sequencing to verify the correctness of the plasmids. 293T with good state and early passage is selected as a lentivirus packaging cell, and the three plasmids are transfected into the 293T cell by using a transfection reagent PEI. Transfection was accomplished in 10cm culture dishes totaling 10mL, and the transfection mixture for each dish of cells should be formulated in 1mL system using serum-free DMEM with the plasmid psPAX 2: plasmid pmd2. g: CJ-PSMA-LIGHT-VTP plasmid: PEI 5 μ g: 3 μ g: 5 μ g: 50 μ l of the transfection mixture was mixed at room temperature, left to stand for 20min and then slowly added to 293T with a cell density of 70-80% in 9mL of the existing medium, and the medium was replaced with fresh medium (DMEM + 10% FBS + 1% P/S) after 6-8 h. And respectively harvesting culture solution supernatants after 48h and 72h of culture, and performing ultrafiltration and super-separation concentration to obtain the virus expressing the chimeric antigen receptor and the LIGHT-VTP, wherein the obtained virus is named as LIGHT-VTP-CAR virus. The CJ-PSMA-CAR plasmid (lacking the second expression cassette for LIGHT-VTP expression compared to the LIGHT-VTP-CAR plasmid) was used as a control, and transfection and treatment were performed according to the above method, and was named PSMA-CAR virus.

Virus titer detection

The method comprises the following steps:

selecting 293T with good state to detect virus titer, inoculating 500 mul of cells with density of 4 x 10^5/mL in a 24-well plate, adding concentrated virus solution with different gradient volumes after the cells are attached to the wall, culturing for 48h, digesting the cells, using CAR to recognize the combined biotinylated PSMA protein, washing after incubating with the cells for 50min at 4 ℃, then using APC-streptavidin SA capable of being combined with biotin to stain for 30min at 4 ℃, washing and loading the cells after staining, using a flow meter to detect CAR positive rate, selecting virus volume with proper positive rate to calculate the virus titer, and using a titer calculation formula: titer (TU/mL) ═ 2X 10⁵X CAR positive rate)/virus volume.

The results of the titer test are shown in FIG. 5, where the titer test was performed according to the above-described titer test method, after the cell plates were attached to the wall, as a controlThe virus PSMA-CAR and the LIGHT-VTP-CAR virus are respectively provided with two volume gradients of 2 mul and 5 mul, in order to avoid false positive caused by nonspecific staining, CTRL is required to be arranged to carry out CAR positive circle gate, and CAR positive cells are obtained when the virus falls into the APC positive circle gate, wherein the proportion value is CAR positive rate. As can be seen from the results of FIG. 5, the 2. mu.l of PSMA-CAR concentrated virus infected with 20 ten thousand 293T can achieve 91.5% positive rate, and the 5. mu.l corresponding positive rate is 97.7%; 2 mul of LIGHT-VTP-CAR virus infected with 20 ten thousand 293T can reach 93.5% of positive rate, 5 mul of corresponding positive rate is 97%, because the positive rate is too high and can not reflect the real titer of the virus, the titer is calculated by using the volume of 2 mul, and the titer of the control virus PSMA-CAR can reach 9.15 multiplied by 10⁷TU/mL, while LIGHT-VTP-CAR virus titer was 9.35X 10⁷TU/mL。

3 construction of chimeric antigen receptor and LIGHT-VTP expressing T cells

The method comprises the following steps:

PBMC is separated from human blood by using lymph separation liquid, then T cells are separated by using a CD4 and CD8 magnetic bead sorting method, after the PBMC is activated by a CD3/CD28 complex for 48 hours, the packed PSMA-CAR and LIGHT-VTP-CAR viruses are used for being infected for 2 hours by centrifugation according to MOI (10), and after 24 hours, the packed PSMA-CAR and LIGHT-VTP-CAR viruses are replaced by a fresh culture medium (XVIVO + 10% FBS + IL-2), and the two CART cells are named as PSMA-CART cells and LIGHT-VTP-CART cells respectively. And after infection and fluid replacement for 48 hours, detecting the CAR expression levels of the two CARTs according to a similar method for titer detection.

The results are shown in FIG. 6, where the CAR positivity of LIGHT-VTP-CART and PSMA-CART is 58.9% and 61.6%, respectively.

LIGHT-VTP expression assay:

for LIGHT-VTP-CAR, not only the expression of CAR needs to be detected, but also whether the CAR has the ability to express LIGHT-VTP needs to be verified, so that supernatants of the two types of CART cells (with consistent positive rate adjustment) under the same culture system condition are collected for 48h, and the expression of LIGHT is verified by ELISA.

The results are shown in FIG. 7, and the results of ELISA showed that LIGHT-VTP-CART cells were at 2X 10⁶The highest LIGHT concentration secreted by each cell per mL can reach 1630pg/mL, while the highest LIGHT concentration secreted by PSMA-CART can reach the highest concentration under the same culture concentration1281pg/mL is achieved, which shows that the embodiment of the invention successfully constructs CAR-T cells over-expressing LIGHT-VTP.

Experimental example 1

LIGHT-VTP enhances the killing effect of CART on tumor cells

LIGHT-VTP-CART and PSMA-CART with consistent CAR-positive rate adjustment were used as effector cells, PC3-PSMA (human prostate cancer cell line, using lentivirus to stably express PSMA and luciferase) was used as target cells, and first, equal amounts of target cells (2 ten thousand) were added to a low-adsorption well plate at an effective-to-target ratio (effector cells: target cells) of 4: 1. 2: 1. 1: 1 corresponding number of CART effector cells were added and wells with different gradients of only target cells (0, 1, 2, 4, 6, 8, 16 ten thousand) were made as standard curves. Because the target cells can express luciferase, after 12h of co-incubation (cc: co-culture), after adding the substrate, the light absorption value is in linear relation with the number of target cells, standard curves can be made, the number of the residual target cells is calculated, thereby calculating the killing efficiency lysine (%) (initial target cell number-remaining target cell number)/initial target cell number, using the killing efficiency lysine (%) as the ordinate and the different effect-to-target ratios (E: T) as the abscissa, the results shown in FIG. 8 were obtained, it can be known that PSMA-CART and LIGHT-VTP-CART have obvious killing effect on human prostate cancer cells, and the killing effect gradually rises with the increase of the effective target ratio, however, the killing effect of LIGHT-VTP-CART on human prostate cancer cells is obviously better than that of PSMA-CART, which shows that CART cells over expressing LIGHT-VTP can enhance the killing capability of the CART cells on tumor cells.

Selecting the following components in percentage by weight E to T as 1: 1 of 12h Co-incubation supernatants was tested for LIGHT expression using ELISA, and the results are shown in FIG. 9, where the antigen-stimulated LIGHT-VTP-CART secreted more LIGHT (at the same culture concentration (1X 10)⁶Individual cell/mL), the LIGHT-VTP-CART secretes LIGHT at a concentration of 423.75pg/mL, the PSMA-CART secretes LIGHT at a concentration of 223.75pg/mL, and the CTRL-T at 68.75pg/mL), further confirming that the effect of LIGHT-VTP enhances the function of LIGHT-VTP-CART.

Experimental example 2

LIGHT-VTP enhances the proliferation of CART

In order to verify the ability of LIGHT to promote T cell proliferation, 20 million LIGHT-VTP-CART and PSMA-CART, each with consistent positive rates, were cultured in the same culture system, and the proliferation of two different CARTs was followed by absolute counting by a counter over two weeks. The results are shown in FIG. 10, where LIGHT-VTP-CART has a significant proliferative advantage.

Experimental example 3

LIGHT combined with CART promotes tumor regression

To preliminarily judge the antitumor effect of LIGHT in combination with CART, experiments were performed in a prostate cancer NSG mouse model using a co-injection of both. The PC3-PSMA cell line (Luciferase marker) was injected subcutaneously into mice, tumor volume was followed using vernier caliper and mouse in vivo imaging techniques, and when a certain size was reached, the following four sets of experiments were set up: PBS, hLIGHT, PSMA-CART + hLIGHT. PSMA-CART was infused only once via the tail vein (3.5X 10)⁶Individual cells/one), PBS was infused only once through the tail vein (100ul PBS/one), hLIGHT (recombinant human full length LIGHT, 100ng/mL) was injected once per week by intratumoral injection, 25ul each time. Post-treatment 2 consecutive tracings by in vivo imaging were performed weekly for 40 days. As shown in FIG. 11 (PSMA-CART (top) and PSMA-CART + hLIGHT (bottom)) groups, it can be seen from the results of in vivo imaging that the combination treatment of CART by tail vein injection and hLIGHT intratumorally effectively reduced the tumor volume (shown as in vivo imaging graph: Radiance (p/sec/cm)²/sr)). FIG. 12 shows the tumor sizes of the mice at day 42, and it can be seen that the tumor volumes of the PSMA-CART + hLIGHT group are significantly smaller than those of the PSMA-CART group.

FIG. 13 shows control group injected with PBS or hLIGHT only, and it can be seen that mice did not get smaller and died at 34 days during follow-up, indicating that hLIGHT alone had no effect on the prostate cancer NSG mouse model (hyperimmune deficiency). Suggesting that LIGHT functions by means of immune cells.

In conclusion, most of the four-stage solid tumors have no or little infiltration of T lymphocytes in the primary tumor when diagnosed, which is why patients have a low response rate to immune checkpoint inhibitors or other immune combination therapies, and the presence of tumor-infiltrating lymphocytes (TILs) in the treatment of various types of cancer is strongly correlated with a good prognosis. Thus TIL is a key factor in immunotherapy, with "hot" tumors referring to tumors that exhibit lymphocyte infiltration and inflammation and "cold" tumors referring to tumors that do not. TIL has become an important scoring criterion and treatment basis in tumor treatment. For solid tumors, which are mostly "cold" tumors, the infiltration of lymphocytes, especially T cells, into the tumor tissue is the first step of the access of CART cells to tumor cells, which is the primary condition for the CART cells to function adequately. The embodiment of the invention designs and constructs a novel 'infiltrative' CART cell which can secrete LIGHT and targets PSMA on the basis of taking the prostate cancer of a typical 'cold' tumor in a solid tumor as an entry point and a second-generation CAR-T technology of targeting PSMA, and the LIGHT is fused with a tumor abnormal blood vessel targeting polypeptide VTP, namely LIGHT-VTP. Therefore, LIGHT-VTP secreted by CAR-T cells can specifically target abnormal tumor blood vessels, thereby promoting the normalization of the abnormal tumor blood vessels, further heating the tumor, promoting the formation of lymphoid structures at the tumor part to guide the CAR-T cells to gather and activate, mobilize autoimmune functions and remodel the inhibitory action of the tumor microenvironment, thereby changing the effect limitation of the CART cells caused by the incapability of penetrating tumor tissues and immunosuppression, enhancing the activity of the CART cells against solid tumors, and greatly improving the effect of the CAR-T in the treatment of the solid tumors such as prostate cancer and the like under the synergistic action of LIGHT-VTP.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of east China

Shanghai Bangyao Biological Technology Co.,Ltd.

<120> enhanced CART cell promoting solid tumor infiltration and preparation method and cell medicine thereof

<130> 250

<160> 17

<170> PatentIn version 3.5

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Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Cys Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

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Thr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly Leu Glu Trp Ile

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Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

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Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

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Val Ser Ser

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gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

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<212> DNA

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ggagagatgg tcacccgcct gcctgacgga cctgcaggct cctgggagca gctgatacaa 60

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aaggtgcagc tgggcggtgt gggctgcccg ctgggcctgg ccagcaccat cacccacggc 300

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<212> PRT

<213> Artificial sequence

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Gly Glu Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp Glu

1 5 10 15

Gln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala His

20 25 30

Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu

35 40 45

Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His

50 55 60

Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser

65 70 75 80

Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser Thr

85 90 95

Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu

100 105 110

Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser

115 120 125

Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu

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Glu Ala Gly Glu Lys Val Val Val Arg Val Leu Asp Glu Arg Leu Val

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Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val

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ggcggcggct gccggggccg gagaagcacc ggc 33

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Gly Gly Gly Cys Arg Gly Arg Arg Ser Thr Gly

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Claims

1. An enhanced CART cell promoting solid tumor infiltration,

the CART cell contains a first nucleic acid sequence and a second nucleic acid sequence;

the second coding sequence is expressed from a constitutive promoter.

2. The solid tumor infiltration-promoting enhanced CART cell according to claim 1, wherein the second nucleic acid sequence is located downstream of the first nucleic acid sequence, the constitutive promoter is located upstream of the first nucleic acid sequence, and both the first coding sequence and the second coding sequence are expressed by the constitutive promoter.

3. The solid tumor infiltration-promoting enhanced CART cell according to claim 1 or 2, wherein the constitutive promoter is selected from the group consisting of EF-1 α, early cytomegalovirus promoter sequence, simian virus 40 early promoter, mouse mammary cancer virus, Human Immunodeficiency Virus (HIV) long terminal repeat promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, rous sarcoma virus promoter, actin promoter, myosin promoter and any of heme promoter and creatine kinase promoter;

preferably, the constitutive promoter is EF-1 α.

4. The enhanced CART cell for promoting infiltration of solid tumors according to claim 1 or 2, wherein the LIGHT protein is a full-length LIGHT protein or a functional fragment thereof;

preferably, the LIGHT protein is a soluble fragment of the LIGHT full-length protein;

preferably, the amino acid sequence of the LIGHT protein is shown as SEQ ID NO. 15.

5. The solid tumor infiltration-promoting enhanced CART cell according to claim 4, wherein the LIGHT protein is fused to a VTP polypeptide;

preferably, the amino acid sequence of the VTP polypeptide is shown as SEQ ID NO. 17.

6. The enhanced CART cell for promoting solid tumor infiltration according to claim 1 or 2, wherein the signal peptide encoded by the signal peptide coding sequence is selected from a membrane-integrated signal peptide or a secreted signal peptide;

preferably, the membrane-integrated signal peptide is selected from any one of a CD8 signal peptide, a CD28 signal peptide, a GM-CSF signal peptide, a CD4 signal peptide and a CD137 signal peptide;

preferably, the secretory signal peptide is selected from any one of an IgG signal peptide and a cytokine signal peptide;

7. The solid tumor infiltration-promoting enhanced CART cell according to claim 1 or 2, wherein the chimeric antigen receptor further has a transmembrane domain and a costimulatory signaling region;

preferably, the transmembrane domain is selected from the transmembrane domains of at least one of the following protein molecules: CD5, CD28, CD137, CD3 epsilon, CD154, CD45, CD4, CD9, CD37, CD16, CD33, CD22, CD134, and CD8 alpha;

preferably, the transmembrane domain is a CD8a transmembrane domain;

preferably, the costimulatory signaling region comprises the intracellular domain of at least one of the following costimulatory molecules: OX40, CD3 γ, CD3 δ, CD134, CD5, CD79a, CD137, ICD3 ε, CD154, CD22, CD66d, CD2, CD28, CD4, CD5, CD79b, COS, 4-1BB, and CD3 ζ;

preferably, the costimulatory signaling region includes the intracellular costimulatory element of 4-1BB and the intracellular domain of CD3 ζ.

8. The enhanced CART cell for promoting infiltration of solid tumors according to claim 1 or 2, wherein the solid tumors are selected from any one of liver cancer, head and neck cancer, melanoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, stomach cancer, bladder cancer, prostate cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma and breast cancer;

preferably, the solid tumor is prostate cancer;

preferably, the antigen binding domain is capable of targeting a specific membrane antigen of prostate cancer, the antigen binding domain is selected from any one of Fab, Fab ', F (ab') 2 and scFv;

preferably, the antigen binding domain is a scFv;

preferably, the amino acid sequence of the light chain variable region of the antigen binding domain is shown as SEQ ID No.3, the amino acid sequence of the heavy chain variable region of the antigen binding domain is shown as SEQ ID No.7, and the amino acid sequence of the hinge region between the heavy chain variable region and the light chain variable region of the antigen binding domain is shown as SEQ ID No. 5.

9. A method of preparing a solid tumor infiltration-promoting enhanced CART cell according to any of claims 1-8, comprising: allowing the target T cell to contain the first nucleic acid sequence and the second nucleic acid sequence.

10. A cell drug for treating solid tumors, comprising the enhanced CART cells for promoting infiltration of solid tumors according to any one of claims 1 to 8 as an active ingredient.